Bone marrow aspirate enhanced bone graft

ABSTRACT

A method of preparing a bone graft. An osteoconductive matrix is placed in a receptacle. Bone marrow aspirate is provided that includes plasma, progenitor cells, hematopoietic cells, endothelial cells, red blood cells, white blood cells, platelets, bone, cartilage, thrombus or combinations thereof. The bone marrow aspirate is passed through the receptacle. At least a portion of bone marrow aspirate is retained in the receptacle. The portion of the bone marrow aspirate retained in the receptacle becomes associated with the osteoconductive matrix to form the bone graft.

REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/517,202, which was filed on Oct. 17, 2104, and this applicationclaims priority to U.S. Provisional Application No. 61/892,756, whichwas filed on Oct. 18, 2013, the contents of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The invention relates generally to bone grafts. More particularly, theinvention relates to bone marrow aspirate enhanced bone graft.

BACKGROUND OF THE INVENTION

In the US, bone grafts are most commonly used in spine fusion surgeryand, more generally, in the fusion or arthrodesis of any skeletal joint.In addition, bone graft is generally used in trauma surgery for thetreatment of fresh fractures and non-unions, which are typicallyidentified as fractures within 6 months that have not healed properly.The bone graft materials typically bridge a gap between bone segmentsand may also provide a three-dimensional scaffold on which the bone cangrow.

Bone graft treatment is also typically used in conjunction with freshfractures where the bone has been shattered or where the patient is at avery high risk of developing a non-union fracture. Because manyfractures are not this severe and can be treated with alternativemethods of fixation, bone grafts are not frequently needed during freshfracture treatments.

Two areas where bone grafts are used is in conjunction with jointreconstruction and joint revision. For example, the bone graft may beused to fill a void between the bone and joint implant in a jointreconstruction surgery.

Joint revision is much more likely to need a bone graft because a largevoid may result from the removal of the original implant. Jointrevisions that use bone graft material therefore usually require arelatively large quantity of the bone graft material.

There are different types of bone graft materials that may be used toassist a patient's body in bone regeneration. These bone graft materialsare typically classified as either natural or synthetic materials.

Natural bone graft materials are classified in the following groups.Autograft is bone graft material that is obtained from the sameindividual that will receive the bone graft material. Allograft is bonegraft material that is obtained from another human source, whichtypically is from cadavers. Xeongraft is bone graft material that isobtained from another species.

Bone grafts can also be categorized by their bone-forming properties asosteoconductive, osteoinductive or osteogenic. Osteoconductivity is theability of a material to provide an appropriate scaffold or matrix uponwhich new bone tissue can form. Osteoinductivity is the ability of amaterial to stimulate the patient's own system to form new bone.Osteogenic material generates new bone tissue itself. Osteoblasts, whichcan be found in bone marrow and mesenchymal cells, are the only cellsthat can create new bone.

Autograft bone has historically been the standard of care because of itsosteoconductive, osteoinductive and osteogenic properties. At the timeof surgery bone is taken from a donor site in the patient, often theiliac crest bone but others are used and then is re-implanted back intothe patient at the surgical site.

Autograft is often not used, because obtaining the graft generallyrequires a second surgical procedure with associated risks and expenses.The autograft also typically results in significant post-operativeissues, most significantly pain. An additional type of autograft,concentrated cells from bodily fluids such as blood or bone marrow, isoften used as well.

In addition to autograft, many other types of bone graft are usedincluding processed cadaver bone, i.e., allograft, in the form ofdemineralized bone matrix and also so called “living cell” or “stemcell” allograft. Additionally, constituents know to be involved in newbone formation, such as bone morphogenic proteins, typically produced byrecombinant processing means, as used. Synthetic materials such astri-calcium phosphate, calcium sulphate, hydroxyapatite and others areused as well.

Summary of Bone Graft Characteristics by Material Type OsteoconductiveOsteoinductive Osteogenic Autograft Yes Yes Yes Bone morphogenic No Yes(strong) Yes proteins Demineralized bone Yes Minimal No matrixAllogeneic stem cell Yes Unknown Yes Bone marrow aspirate Yes Yes(strong) Yes Synthetics Yes No No

Bone graft substitutes also fall within the classification of bonefiller materials. Examples of bone graft substitutes include collagen,polymers such as silicone and some acrylics, hydroxyapatite, calciumsulfate and ceramics.

Bone cement (such as polymethylmethacylate) can be used as a bone voidfiller to treat bone voids or defects. For example, it can be used torepair fractured bones and vertebral bodies. The bone cement can be usedeither in procedures that involve direct injection of the bone cementinto the fractured vertebral body (i.e., vertebroplasty) or injection ofthe bone cement into the vertebral body after the height of thevertebral body is restored using a pressurized balloon (i.e.,kyphoplasty).

One of the disadvantages of using bone cement is that, once it isinjected inside the patient, the bone cement is an inorganic materialthat acts as a foreign body. As such, the bone cement may not onlynegatively impact healing but can also lead to bone disease.

Additionally, the bone cement is typically stiffer than bone, which mayincrease the incidence of adjacent level fractures in the spine. Bonecement leakage may cause complications, and has been reported to occurin vetebroplasty and kyphoplasty procedures. If leakage does occur, thebone cement can cause soft tissue injury due to the high temperatures ofthe exothermic polymerization reaction. In addition, if the bone cementis forced into the vascular system, it can cause emboli.

Bone marrow and bone marrow aspirate concentrate is considered to have asignificantly higher bioactivity than circulating blood or concentratedblood known as platelet rich plasma and is preferred for use inorthopedic applications because it contains progenitor cells andmultipotent stem cells, which assist in the formation of new bone.

Traditionally, doctors have used a large bore needle to aspirate bonemarrow. However, orthopedic companies have developed their own versionsof bone marrow aspirate concentrate systems for use specifically withbone graft substitute. These disposable kits are used for aspirating andconcentrating the stem cells found in the bone marrow onto a graftmatrix to be implanted into the patient. Combined with a bone graftsubstitute, bone marrow aspirate concentrate may provide similar resultsto an autograft (Geistlich, 2011).

Bone marrow aspirate concentrate has become increasingly popular in bonegrowth applications, particularly spinal fusion and trauma surgery,because of its osteogenic properties. Traditionally, autograft was thegold standard grafting material in these procedures due to the presenceof osteoblasts and osteogenic precursor cells, as well as itsosteoconductive and osteoinductive properties.

To avoid the risks associated with autograft procurement such as donorsite infection and morbidity, bone marrow aspirate concentrate has beenincreasingly used because it has similar properties as autograft andallows surgeons and patients to avoid autograft procurement.

Muschler, U.S. Pat. Nos. 5,824,084 and 6,049,026, are directed to amethod and apparatus for preparing composite bone graft. Demineralizedbone is placed in a receptacle having an inflow opening and an outflowopening. Bone marrow aspirate is introduced to the receptacle throughthe inflow opening.

Muschler indicates that the matrix material has a surface thatselectively bonds to progenitor cells to retain the progenitor cells inthe matrix. The porous nature of the matrix material allows other cellssuch as blood cells to pass through.

Muschler, U.S. Pat. No. 6,723,131, describes placing a biocompatibleimplantable matrix such as demineralized bone in a cartridge. Bonemarrow aspirate is placed into a syringe that is connected to an inputport on the cartridge. A collection syringe is attached to an outletport on the cartridge to withdraw material that does not adhere to thematrix. It is indicated that the invention provides an enrichedpopulation of progenitor cells.

Merboth et al., U.S. Pat. No. 7,018,382, discloses a bone marrow mixinginstrument. The instrument includes one tube for demineralized bone andone tube for bone marrow aspirate. Both of the tubes are connected to atrigger mechanism that causes the demineralized bone and the bone marrowaspirate to be discharged at the desired respective rates.

Behnam et al., U.S. Patent Publication No. 2009/0155378, describestreating bone marrow aspirate to increase osteoinductive activity.Behnam indicates that bone marrow aspirate may be mixed with osteoplant.

Rosenberg et al., U.S. Pat. No. 7,582,309, describes forming cohesivedemineralized bone compositions with elongated demineralized bone fibersand a biocompatible liquid. Rosenberg indicates that the elongateddemineralized bone fibers may be formed using a shaving process.

Bays et al., U.S. Pat. No. 7,776,594, discloses placing demineralizedbone in a cartridge. Bone marrow aspirate is then flowed through thecartridge. A filter is placed at a distal end of the cartridge thatenables air to pass therethrough but prevents any of the components inbone marrow aspirate from passing therethrough.

Huang et al., U.S. Patent Publication No. 2012/0251609, describes ademineralized cancellous bone matrix having a calcium concentration ofbetween about 5% and 20% and where one of the dimensions of thedemineralized cancellous bone matrix is between 0.5 millimeters andabout 20 millimeters.

SUMMARY OF THE INVENTION

An embodiment of the invention is directed to a method of preparing abone graft. An osteoconductive matrix is placed in a receptacle. Bonemarrow aspirate is provided that includes plasma, progenitor cells,hematopoietic cells, endothelial cells, red blood cells, white bloodcells, platelets, bone, cartilage, thrombus or combinations thereof. Thebone marrow aspirate is passed through the receptacle. At least aportion of bone marrow aspirate is retained in the receptacle. Theportion of the bone marrow aspirate retained in the receptacle becomesassociated with the osteoconductive matrix to form the bone graft.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of embodiments and are incorporated in and constitute apart of this specification. The drawings illustrate embodiments andtogether with the description serve to explain principles ofembodiments. Other embodiments and many of the intended advantages ofembodiments will be readily appreciated as they become better understoodby reference to the following detailed description. The elements of thedrawings are not necessarily to scale relative to each other. Likereference numerals designate corresponding similar parts.

FIG. 1 is a side view of collecting bone marrow aspirate in a collectionvessel.

FIG. 2 is a side view showing addition of an aggregating agent to thebone marrow aspirate in the collection vessel.

FIG. 3 is a side view showing red blood cells precipitated from the bonemarrow aspirate in the collection vessel.

FIG. 4 is a side view showing the precipitated red blood cells beingremoved from the collection vessel.

FIG. 5 is a side view showing bone marrow aspirate being passed from thecollection vessel and through a graft container.

FIG. 6 is a sectional view of the graft container.

FIG. 7 is a side view showing the graft container being opened to removethe graft.

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the invention is directed to bone marrow aspirateenhanced bone graft. The bone graft thereby includes bone marrowaspirate and demineralized bone matrix.

Bone marrow aspirate contains plasma, progenitor cells, hematopoieticcells, endothelial cells, and cells derived from peripheral blood, suchas red blood cells, white blood cells and platelets.

Bone marrow aspirate may be obtained from a source that minimizes thepotential of immunological reactions in the person who is receiving thebone graft. As such, it is desirable for the bone marrow aspirate to beobtained from a person who is determined to be immunologicallycompatible with the bone graft recipient. In certain embodiments, bonemarrow aspirate is obtained from the patient who will receive the graft.

Because the beneficial constituents in bone marrow aspirate represent avery small percentage of the overall volume of the bone marrow aspirate,it is desired to concentrate the bone marrow aspirate to reduce thevolume of non-beneficial fractions including water and red blood cells.It is also desired to remove a significant portion of the red bloodcells from the bone marrow aspirate as part of the invention because redblood cells are known to be inflammatory and, therefore, can impede thebone healing process.

The standard practice for concentrating bone marrow aspirate into bonemarrow aspirate concentrate involves use of a centrifuge, which relieson density differences to separate the constituents of bone marrowaspirate. Unfortunately, red blood cells may be captured in the samedensity gradient as the desirable cells and constituents. Therefore,centrifugation is less than an optimal technique for separating the redblood cells from the other components of the bone marrow aspirate.Additionally, the centrifugation forces can damage the red blood cells,which could make it more difficult to separate the red blood cells fromthe remainder of the bone marrow aspirate.

Red blood cells carry a strong negative charge and, therefore, can beattracted to a positive charge and thereby clump together into a mass ofsufficient density to precipitate out of solution.

This invention uses a reagent that utilizes the strong negative chargeof the red blood cells to cause aggregation of the red blood cells. Thereagent causes the red blood cells to bind together by means of electriccharge attraction. Once the red blood cells have been aggregated, theaggregated red blood cells can be precipitated out of bone marrowaspirate.

The reagent having the positive charge should efficiently attract thered blood cells while facilitating separation of a large portion of theresultant product from the bone marrow aspirate.

As used herein, the term “large portion” means that more than about 90percent by weight of the resultant product is separate from the bonemarrow aspirate. In other embodiments, the term “large portion” meansthat more than about 95 percent by weight of the resultant product isseparated from the bone marrow aspirate.

The positively charged material should also be relatively inert withrespect to the other desirable components in the bone marrow aspiratesuch that the positively charged material does not impact the beneficialproperties of the bone marrow aspirate.

Furthermore, the positively charged material should have no negativeinteractions if any of the positively charged material remains in thebone marrow aspirate, which then becomes incorporated into the bonegraft and thereafter is implanted into the patient.

An example of one such positively charged material that may be used inconjunction with separating the red blood cells from the bone marrowaspirate is marketed under the designation PrepaCyte by BioE LLC fromSt. Paul, Minn. USA.

An alternative to utilizing the negatively charged characteristics toseparate the red blood cells from the other portions of the bone marrowaspirate utilizes chemically reacting the red blood cells with anothercompound. The resulting product is more dense than the other portions ofthe bone marrow aspirate, which facilitates separation of the resultingproduct from the remainder of the bone marrow aspirate.

An example of one technique that may be used to separate the resultantproduct from the bone marrow aspirate is gravity separation. Because ofthe agglomeration of the charged or reacted components, the resultantproduct has sufficient size such that it settles to the bottom of thebone marrow aspirate.

In certain embodiments, the mixing of the bone marrow aspirate and thepositively charged material may be done in a syringe. After theresultant product has settled in the syringe, the syringe may beoriented so that the syringe outlet is pointed upwardly and thesupernatant can be discharged from the syringe.

The supernatant contains all of the desirable constituents present inbone marrow aspirate. To facilitate further processing of thesupernatant for use in the bone graft material, the supernatant can becollected in another syringe.

Alternatively or additionally to utilizing the syringe, the separationcan be facilitated using a centrifuge. If centrifugation is used, itshould be done in a manner that minimizes disruption of the agglomeratesas well as to minimize lysing of the red blood cells.

In addition to facilitating removal of red blood cells, another benefitof this reagent method is that the cells present in the supernatant aresubstantially unaffected and undamaged in the process of red blood cellaggregation and precipitation.

In another embodiment of the invention, the bone marrow aspirate isconcentrated prior to being mixed with the demineralized bone matrix.Such separation technique should facilitate a large portion of the waterin the bone marrow aspirate to be separated from the other portions ofthe bone marrow aspirate.

One technique that may be used in the concentration process iscentrifugation. Use of a centrifuge is effective at cell concentrationbut is less desirable in the environment intended for this invention.

A centrifuge is a fairly large mechanical apparatus intended forlaboratory use and may present challenges to be safely used in anoperating room. In addition, the centrifuges are bulky thereby taking upconsiderable table space, noisy and relatively slow.

The intended environment for this system is the sterile operating fieldinside a hospital operating room at the time of surgery, i.e., at thepoint and time of use. Every time the centrifuge is used in the sterilefield of an operating room, the centrifuge needs to be sterilized. Thecentrifuge needs to be accessible and useable in a manner that maintainssterility of not only the bone marrow aspirate but also the area inwhich the surgical procedure is being performed.

Another option for concentrating the bone marrow aspirate in a sterileenvironment utilizes a filter that is selected with an appropriate portsize that permits water in the bone marrow aspirate to pass therethroughwhile preventing a significant portion of the other components in thebone marrow aspirate from passing therethrough. The filter may be amembrane type, similar to a coffee filter where a relatively thinmembrane with tightly controlled porosity serves to allow passage ofliquid but retention of solids larger than the pore size of themembrane.

Alternatively, the filter may consist of a bed or column of porousmaterial such as granules or particles but may also be a porousmonolithic structure such as a sponge. In the case of a filter bed orcolumn, modulation of particle or pore size, packing density and aspectratio of the column can be used to establish and control the effectiveporosity and therefore filtering specificity and efficiency. In certaincases a gradation of granule or particle size, such as from larger tosmaller, can be used improve filter efficiency and specificity. A graveland sand water filter is an example.

Trapping can occur by means of mechanical separation, i.e., cells thatare too large to pass through the porous network of the filter willbecome lodged in the network. Alternatively the trapping can utilizeadsorption to the surface of the filter components, i.e., chemical,electrical attraction, van der waals forces, covalent bonds, hydrogenbonds, electrostatic interactions, etc.

Affinity chromatography is an example of a means for trapping selectconstituents from a solution wherein a sorbent, such as demineralizedbone matrix and certain extracellular matrix materials such as collagen,keratin sulfate, heparin, etc. are used to selectively attract andretain desirable constituents.

A suitable membrane is available under the classification of micropore.Using such a membrane causes substantially all of the components in thebone marrow aspirate to be retained on the surface of the filter mediumwhile the water molecules pass through the filter medium.

To enhance the amount of water that is removed from the bone marrowaspirate, a reduced pressure may be utilized on the side of the filtermedium that is opposite the bone marrow aspirate. The amount of thereduced pressure that is needed may be affected by factors such as thepore size of the filter medium and the desired water concentration ofthe concentrated bone marrow aspirate.

The membrane may be formed with a relatively large surface area ascompared to a diameter of a conduit that is used to deliver the bonemarrow aspirate to the filter medium. Forming the filter medium with arelatively large surface area enables the bone marrow aspirate to bedispersed in a relatively thin layer.

This relatively thin layer not only reduces the time for the water to beseparate from the other portions of the bone marrow aspirate but alsoreduces the force that must be applied to the bone marrow aspirateduring the filtration process, which reduces the potential of damage tothe cells in the bone marrow aspirate.

Thereafter, the concentrated bone marrow aspirate is removed from thefilter medium. An example of one suitable technique involvesmechanically scraping the concentrated bone marrow aspirate. Care shouldbe exercised to minimize not only damage to the cells in the bone marrowconcentrate but also to avoid damage to the filter medium.

Another technique that may be used to remove the concentrated bonemarrow aspirate from the filter medium is with a liquid such as water.The liquid may facilitate separation of the concentrated bone marrowaspirate from the filter medium with less damage than the mechanicalscraping.

The volume of water used to separate the concentrated bone marrowaspirate from the filter medium should be as small as possible toachieve separation of the concentrated bone marrow aspirate from thefilter medium while enabling the concentrated bone marrow aspirate to beas concentrated as possible.

In certain embodiments, the liquid may be provided from a side of thefilter medium that is opposite the side of the filter medium on whichthe bone marrow aspirate has accumulated. In other embodiments, theliquid may be provided from the side of the filter medium on which thebone marrow aspirate has accumulated.

One challenge with forming the filter medium with a relatively largesurface area as compared to a diameter of the conduit that is used todeliver the bone marrow aspirate to the filter medium is that dependingon the flow rate of the bone marrow aspirate, only a portion of thefilter medium may actually be used.

To enhance the efficiency of the water removal from the bone marrowconcentrate, a multiple stage filter can be used. Each of the filtermediums in the multiple stage filter may have a smaller surface areathan the single stage filter that is discussed above. The filter mediaused in the different stages may be selected with different pore size.In one such configuration, the pore size of the successive filter mediahas a progressively smaller pore size.

Regardless of concentration means, the desirable constituent fractionharvestable from a quantity of bone marrow aspirate taken from a patientwill be a fairly small volume of material. In some embodiments, theamount of bone marrow aspirate is between about 1 cm³ and about 3 cm³.

In addition to the advantageous components in the bone marrow aspirateconcentrate, it is desired to provide a cell binding and cell friendly,osteoconductive matrix to facilitate use of the invention in providing astrong bone graft.

The osteoconductive matrix may include a combination of demineralizedbone matrix, a suitable synthetic alternative such as hydroxyapatitewith the addition of other materials that fall within the classificationof extracellular matrix. Examples of these materials include hyaluronicacid, collagen, keratin, elastin, fibronectin and laminin.

The osteoconductive matrix can be configured as a filter for selectiveretention of the desirable constituents of red blood cell depleted bonemarrow aspirate supernatant whereby the osteoconductive matrix filtersthe desirable constituents by means of mechanical filtering such as bycontrolled porosity and/or by means of selective surface binding such asaffinity chromatography like effect.

In certain embodiments, the combination includes greater than about 50%by weight demineralized bone matrix or synthetic substitute thereof. Inother embodiments, the combination includes demineralized bone matrix orsynthetic substitute thereof at a concentration of between about 60% and90% by weight.

The demineralized bone matrix that is used in preparing the bone graftmay be provided in a variety of forms such as powder, small particles orin the shape of the implant. The demineralized bone matrix can beobtained from various commercial sources such as AlloSource, Cryolife orRTI Biologics.

In certain embodiments, the demineralized bone matrix may have anaverage particle size of less than about 1 millimeter. In otherembodiments, the demineralized bone matrix may have an average particlesize of less than about 0.5 millimeters.

Because of the intended in-vivo use of the bone graft, the demineralizedbone matrix should be provided in a sterile configuration to minimizethe potential of introducing pathogens during the process of implantingthe bone graft.

Prior to using the bone graft, the components used to fabricate the bonegraft should be relatively uniformly mixed. A hollow cartridge may beused to mix the red blood cell depleted bone marrow aspirate concentrateand the filtering osteoconductive matrix, e.g., a demineralizedbone/extracellular matrix composite. In certain embodiments, thecartridge may have a generally cylindrical shape with an opening thatextends between opposite ends thereof.

The interior volume of the cartridge may be selected based upon theamount of bone graft that is desired to be produced. In certainembodiments, the demineralized bone matrix may substantially fill theinterior of the cartridge when initially placed therein.

A cover may be provided at opposite ends of the cartridge. The coversmay be removably attached to the cartridge. The attachment of the coversto the cartridge should resist the passage of water therethrough. Avariety of techniques may be used to removably attach the covers to thecartridge. An example of one suitable technique for attaching the coversto the cartridge is a plurality of threads.

The covers would be attached to the cartridge during the process ofmixing the bone marrow aspirate concentrate with the demineralized bonematrix. After the components are mixed, the covers may be detached fromthe cartridge to facilitate removing the bone graft from the cartridge.

The first cover may have an opening formed therein. The openingfacilitates delivering the bone marrow aspirate concentrate to thecartridge. Proximate the opening, a connector may be provided tofacilitate attaching the syringe in which the bone marrow aspirateconcentrate is stored to the first cover.

The connection mechanism may be designed to be substantially water tightand facilitate transfer of the bone marrow aspirate concentrate betweenthe syringe and the cartridge in a sterile manner. An example of onesuitable connection mechanism is a leur lock. An alternative connectionmechanism may be utilized a friction fit between the syringe and thecover.

The second cover may have an opening formed therein. This openingfacilitates drainage of the water that passes through the cartridge.This opening may be connected to a drain line or to a drain storagecontainer.

The filtering matrix that is placed in the cartridge can be allogeneic,synthetic or a combination thereof. The allogeneic material can beprovided in a variety of forms. Examples of two such suitable forms aregranules and fibers.

The allograft can be provided as mineralized or demineralized dependingon the intended use of the graft. In certain embodiments, the allograftgranules have a particle size that is between about 3 millimeters andabout 100 microns.

Examples of the synthetic materials include calcium phosphate,tri-calcium phosphate, hydroxyapatite or combinations thereof. Thesynthetic materials may be provided in a variety of particle sizes suchas between about 3,000 microns and 60 microns.

The bone matrix may be compacted when being placed in the cartridge toenhance the uniformity at which the bone marrow aspirate concentratewill pass through the demineralized bone matrix.

In addition to using a chromatography effect to selectively retain theefficacious components in the red blood cell depleted bone marrowaspirate in the filtering osteoconductive matrix, e.g., a demineralizedbone plus extracellular matrix composite, it is also possible to use amechanical entrapment or filtering effect to selectively retain theefficacious components in the bone marrow aspirate in the demineralizedbone matrix.

The filtering matrix may have a multi-strata configuration. In certainembodiments, the filtering matrix is configured to go from high inherentporosity proximate the inlet to lower inherent porosity proximate theoutlet. Alternatively, it may be possible to use a reverse descendingstrata configuration to help keep the smallest particles in place.

For example, a more coarsely ground demineralized bone matrix is placedproximate the entry port of the cartridge and more finely grounddemineralized bone matrix is placed proximate the outlet port of thecartridge. It is also possible to put one or more additional layersbetween the more coarsely ground layer and the more finely ground layerthat progressively include more finely ground particles.

The bone matrix may consist of a thin layer of 250+ micron particlesthat is placed on the membrane. Next, a slightly thicker layer of 100+micron particles is placed in the cartridge. This process is repeatedwith 250+ micron particles, 500+ micron particles, 1,000+ micronparticles and 3,000+ micron particles.

The effective porosity of a bed of granules is between about 25 percentand about 30 percent of the granule size. For example, a bed of 100micron granules will exhibit an effective porosity of about 25 micronsto about 30 microns. The typical granule size range of sieved particlescan be less than 40 microns, between 60 microns and 100 microns, between100 microns and 250 microns, between 250 microns and 500 microns,between 500 microns and 1,000 microns, between 1,000 microns and 3,000microns and greater than 3,000 microns.

The bone marrow aspirate or supernatant will be introduced at the top ofthe cartridge and then pass through the 3,000 micron layer and then the1,000 micron layer and so on. The larger constituents that may be in thebone marrow aspirate such as small pieces of bone, cartilage or thrombuswill be trapped in the first layer but the smaller constituents willpass through all the way down to the stem cell size, which will betrapped in the 60 micron layer or the 100 micron layers as the size ofthese cells is in the range of 15 to 50 microns.

A filter membrane can be provided proximate the outlet port of thecartridge that retains the bone matrix in the cartridge. In certainembodiments, the membrane has a particle size of about 250 microns. Themembrane will retain the particles having a size of greater than 250microns. The 250 micron layer will retain the 100 micron particles. The100 micron layer will retain the 60 micron particles.

It is also possible to utilize an affinity mechanism to trap desirablecells in the bone matrix. This process may be a preferred retentionmechanism as in certain configurations, it is more discriminating forthe preferred cells rather than just size. A person of skill in the artwill appreciate that a variety of compositions may be utilized basedupon the cells that are desired to be trapped in the bone matrix. Forexample, the attractant composition may exhibit a charge that causes thedesired cells to be attracted to the bone matrix.

This attractant composition can be mixed with the bone matrix before thebone matrix is placed into the cartridge. Alternatively, attractantcomposition can be passed through the bone matrix after the bone matrixis placed in the cartridge.

The second syringe having the bone marrow aspirate concentrate placedtherein is connected to the inlet port on the cartridge. A force is thenapplied to the second syringe to cause the bone marrow aspirateconcentrate to move from the second syringe to the cartridge.

To minimize the potential of a channel being formed through thedemineralized bone matrix in the cartridge, the rate at which the bonemarrow aspirate concentrate is ejected from the second syringe shouldnot be too fast.

Another factor believed to play an important role in distribution of thebone marrow aspirate concentrate throughout the demineralized bonematrix is the use of a relatively consistent pressure to cause the bonemarrow aspirate concentrate to be ejected from the second syringe.

The force to cause the bone marrow aspirate concentrate to be ejectedfrom the second syringe may be applied by a person's hand or hands. Ifthe force is applied by the person's hand or hands, guidance should beprovided regarding the total time for all of the bone marrow aspirateconcentrate to be ejected from the second syringe.

Alternatively, the second syringe may be placed in a mechanical ejectorthat is capable of ejecting the bone marrow aspirate concentrate fromthe second syringe at approximately the desired rate. In one suchconfiguration, the mechanical ejector is a spring. In other embodiments,the ejector is operated with hydraulic pressure, pneumatic pressureand/or pressure applied by a motor.

Proximate a second end of the cartridge that is opposite the end of thecartridge to which the second syringe is attached, a filter membrane maybe placed. The filter membrane may be formed with a pore size that issufficiently small such that it resists passage of the demineralizedbone matrix and all of the components in the bone marrow aspirateconcentrate except water. The filter membrane may also permit air andother gases that are present in the demineralized bone matrix and/or thebone marrow aspirate concentrate to pass therethrough.

Allowing the water to pass through the filter membrane facilitatesfurther concentration of the bone marrow aspirate concentrate during theprocess of intermixing the bone marrow aspirate concentrate and thedemineralized bone matrix.

The size and/or number of pores in the filter membrane can also be usedto control the amount of water that passes through the filter membraneand, as such, can adjust the moisture level of the bone paste that isprepared from the intermixing of the bone marrow aspirate concentrateand the demineralized bone matrix.

While it is possible to attach a separate filter to the second end ofthe cartridge, such a configuration would enable a portion of the bonemarrow aspirate concentrate and the demineralized bone to exit from thesecond end of the cartridge. Since the opening in the second end of thecartridge has a diameter that is considerably smaller than the diameterof the cartridge, the bone marrow aspirate concentrate and thedemineralized bone matrix may preclude the flow of water out of thesecond end of the cartridge. Accordingly, it may be desirable to preventthe bone marrow aspirate concentrate and the demineralized bone matrixfrom exiting the second end of the cartridge.

A person of skill in the art can appreciate that a variety of porousmaterials can be used to prevent the bone marrow aspirate concentrateand the demineralized bone matrix from exiting the second end of thecartridge. An example of one such porous material is filter media havinga pore size that is smaller than the pore size of the components of thebone marrow aspirate concentrate and the demineralized bone matrix.

After all of bone marrow aspirate concentrate has been ejected from thesecond syringe into the cartridge, the bone marrow aspirate concentrateand the demineralized bone matrix may be sufficiently well mixed suchthat the resulting bone paste is ready for use. In other situations,additional mixing may be desired to ensure that the bone paste isrelatively uniformly mixed.

One option for applying the bone paste is to remove at least one of thecovers from the cartridge and then scoop or otherwise remove the bonepaste from the cartridge. In another configuration, the first cover isremoved and replaced with a plunger mechanism and the second cover isremoved and replaced with a dispensing tip. This process therebyconverts the cartridge into a syringe such that the plunger can be usedto dispense the bone paste from the cartridge in a controlled manner.

Depending on the desired application of the bone paste, the bone pastecan be formed with different flowabilities. The flowability of the bonepaste can be adjusted by changing the amount of water in the bone marrowaspirate concentrate as well as the amount of water that is allowed topass through the filter membrane at the second end of the cartridge.

Alternatively to applying the bone graft as a paste, it is possible toform the bone graft material into the desired shape of the implant.After the bone graft material is formed into the desired shape, themoisture content of the bone graft material can be reduced to cause theimplant to become more rigid. An example of one technique that may beused to reduce the moisture content of the implant is heating.

The physical properties of the bone graft material may be enhanced bythe addition of at least one additive to the bone graft material. Anexample of one additive is collagen.

In another embodiment of the invention, the demineralized bone matrix isformed with a larger particle size. The bone marrow aspirate concentratemay flow through the cartridge more than one time to provide thecomponents in the bone marrow aspirate concentrate with a greateropportunity to associate with the demineralized bone matrix particles.

The product and method of the present invention are described in thefollowing examples. These examples are provided as an illustration ofthe invention and are not intended to limit the invention.

EXAMPLE

As an initial aspect in performing the method of this invention, bonemarrow is obtained from a person. A person of skill in the art willappreciated that a variety of techniques may be used to obtain the bonemarrow. As noted above, it may be possible for the bone marrow to beobtained from the same person on which it is intended to use the bonegraft prepared therefrom. In other situations, the bone marrow isobtained from a person who is immunologically compatible with the personin which it is intended to use the bone graft prepared therefrom.

The bone marrow aspirate 10 is placed in a collection vessel 12 thathaving a generally cylindrical shape, as illustrated in FIG. 1. A firstopening 14 is provided proximate an upper end of the collection vessel12. It may be possible to attach a first cover 16 the first opening 14.The first cover 16 includes an inlet port 20 and an outlet port 22. Thefirst cover 16 may be removably attached to the collection vessel 12such as using complementarily shaped threads on the collection vessel 12and the first cover 16.

A second opening 24 is provided proximate a lower end of the collectionvessel 12. The second opening 24 may have a size that is smaller thanthe first opening 14. Forming the second opening 24 with a smaller sizeenhances the ability to separate the precipitated red blood cells fromthe bone marrow aspirate while minimizing the loss of the other portionsof the bone marrow aspirate, which is described in more detail herein.

In certain embodiments, a size of the second opening 24 is less thanabout 25 percent of a size of the first opening 14. In otherembodiments, the side of the second opening 24 is less than about 10percent of the size of the first opening 14.

A second cover 26 is attached to the second opening 24 to therebycontrol the discharge of product from the second opening 24. In certainembodiments, the second cover 26 is removably attached to the collectionvessel 12 such as using complementary shaped threads on the collectionvessel 12 and the second cover 26. While not illustrated, the secondcover 26 may comprise a valve that allows the flow of material from thecollection vessel 12 to be controlled.

The aggregating agent 30 is then added to the collection vessel 12, asillustrated in FIG. 2. The aggregating agent 30 causes red blood cellsin the bone marrow aspirate to agglomerate and then precipitate frombone marrow aspirate, as illustrated at 31 in FIG. 3. The aggregationand settling was substantially complete over a period of time of about25 minutes. Such precipitation facilitates separation of the red bloodcells from the other portions of the bone marrow aspirate.

Next, the precipitated red blood cells are separated from the otherportions of the bone marrow aspirate. In certain embodiments, the firstcover 16 is detached from the collection vessel 12 and a plunger 28 isattached to the collection vessel 12 proximate the first opening 14. Theplunger 28 urges the precipitated red blood cells from the collectionvessel 12 through the second opening 24. The precipitated red bloodcells that are urged from the collection vessel 12 may be discarded.This process was done slowly to minimize loss of the bone marrowaspirate other than the red blood cells.

A graft filter chamber 32 is attached to the second opening 24, asillustrated in FIG. 5. The graft filter chamber 32 has an opening 34formed therein, as illustrated in FIG. 6. The opening 34 is filled withbone graft material 36. In certain embodiments, the bone graft material36 is provided in at least two granulations. More coarsely ground bonegraft material 36 is placed proximate a proximal end of the graft filterchamber 32 and more finely ground bone graft material 36 is positionedproximate a distal end of the graft filter chamber 32, as illustrated inFIG. 6. The graft filter chamber 32 may include at least two sectionsthat are detachable from each other after the process is completed toremove the graft therefrom.

Suction was applied to the distal end of the graft filter chamber 32 tocause the bone marrow aspirate to be drawn through the bone graftmaterial 36. Alternatively or additionally, the bone marrow aspirate maybe urged through the graft filter chamber 32 such as using a plungersimilar to illustrated in FIG. 4. The bone graft material 36 acts as afilter to cause beneficial components in the bone marrow aspirate to beretained in the graft filter chamber 32.

The graft filter chamber 32 is opened as illustrated in FIG. 7 to accessthe bone graft material 36. In certain embodiments, the bone graftmaterial may be used upon formation. In other embodiments, the bonegraft material is stored prior to use. A person of skill in the art willappreciate the storage conditions for the bone graft material tomaintain the efficacy of the bone graft material.

It is to be understood that other embodiments may be utilized andstructural or logical changes may be made without departing from thescope of the present invention. The preceding detailed description,therefore, is not to be taken in a limiting sense, and the scope of thepresent invention is defined by the appended claims.

It is contemplated that features disclosed in this application, as wellas those described in the above applications incorporated by reference,can be mixed and matched to suit particular circumstances. Various othermodifications and changes will be apparent to those of ordinary skill.

1. A method of preparing a bone graft comprising: placing anosteoconductive matrix in a receptacle, providing bone marrow aspiratecomprising plasma, progenitor cells, hematopoietic cells, endothelialcells, red blood cells, white blood cells, platelets, bone, cartilage,thrombus or combinations thereof; passing the bone marrow aspiratethrough the receptacle; and retaining at least a portion of bone marrowaspirate in the receptacle, wherein the portion of the bone marrowaspirate retained in the receptacle becomes associated with theosteoconductive matrix to form the bone graft.
 2. The method of claim 1,wherein an affinity mechanism between the osteoconductive matrix and thebone marrow aspirate causes the portion of the bone marrow aspirate tobe retained in the receptacle.
 3. The method of claim 1, whereinmechanical entrapment causes the portion of the bone marrow aspirate tobe retained in the receptacle.
 4. The method of claim 1, and furthercomprising removing a significant portion of the red blood cells fromthe bone marrow aspirate before passing the bone marrow aspirate throughthe receptacle.
 5. The method of claim 1, and further comprisingconcentrating the bone marrow aspirate before passing the bone marrowaspirate through the receptacle.
 6. The method of claim 1, wherein theosteoconductive matrix is placed in the receptacle in a multi-strataconfiguration in which the osteoconductive matrix has different particlesize regions.
 7. The method of claim 1, and further comprising placing amembrane in the receptacle to substantially retain the osteoconductivematrix in the receptacle as the bone marrow aspirate is passed throughthe receptacle.
 9. The method of claim 1, wherein a substantiallyconstant pressure is used to urge the bone marrow aspirate through theosteoconductive matrix to substantially uniformly distribute the bonemarrow aspirate in the osteoconductive matrix.
 10. The method of claim1, and further comprising controlling flow of water from the outlet portto provide the bone graft with a viscosity that is within a selectedrange.
 11. The method of claim 1, wherein the osteoconductive matrixcomprises a demineralized bone matrix, hydroxyapatite, hyaluronic acid,collagen, keratin, elastin, fibronectin, laminin or combination thereof